Protein Extraction and Western Blot Analysis

Cells were scraped into ice-cold phosphate buffered saline (PBS) containing 1x Sigma Fast Protease Inhibitor Cocktail (Roche) and collected as a pellet by centrifugation (500xg, 5 min, 4°C). Cells were lysed (45 min., 4°C) by agitation in modified RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2% NP-40, 0.25% Deoxycholate, 0.1% SDS, 1mM NaF, 1mM DTT, 1X SigmaFast, 10% glycerol), followed by centrifugation at 20,000xg for 30 min. at 4°C and determination of the total protein concentration in each lysate using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Equal amounts of each lysate (50 μg) were fractionated by 8% PAGE, transferred to nitrocellulose membrane and blocked with 0.1% Casein in 0.2x PBS for 1 hour at room temperature. Membranes were incubated with protein-specific antibodies overnight at 4°C followed by 1 hour treatment with secondary antibodies prior to quantification using chemiluminescence or LiCOR Odyssey. Antibodies used are as follows: anti-LDAH (described previously^{14 (link)}); anti-GFP (NB600–308) from Novus Biologicals, Centennials, CO; anti-Calnexin (ADI-SPA-865-D) from Enzo Life Sciences, Farmingdale, NY; anti-ATGL (2138S) from Cell Signaling Technology, Danvers, MA; anti-αTubulin (T6199) from Sigma-Aldrich, St. Louis, MO.